TGF.beta.
The transforming growth factor -.beta. (TGF.beta.) polypeptides influence growth, differentiation, and gene expression in many cell types. The first polypeptide of this family that was characterized, TGF.beta.1 has two identical 112 amino acid subunits which are covalently linked. TGF.beta.1 is a highly conserved protein with only a single amino acid difference distinguishing human from mice forms. There are two other members of the TGF.beta. gene family that are expressed in mammals. TGF.beta.2 is 71% homologous to TGF.beta.1(de Martin et al., (1987) EMBO J. 6:3673-3677), whereas TGF.beta.3 is 80% homologous to TGF.beta.1 (Derynck et al., (1988) EMBO J 7:3737-3743). The structural characteristics of TGF.beta.1 as determined by nuclear magnetic resonance (Archer et al., (1993) Biochemistry 32:1164-1171) agree with the crystal structure of TGF.beta.2 (Daopin et al., (1992) Science 257:369-374; Schlunegger and Grutter (1992) Nature 38:430-434).
Even though the TGF.beta.'s have similar three dimensional structures, they are by no means physiologically equivalent. There are at least three different extracellular receptors, type I, II and III, involved in transmembrane signaling of TGF.beta. to cells carrying the receptors. For reviews, see Derynck (1994) TIBS 19:548-553 and Massague (1990) Annu. Rev. Cell Biol 6:597-641. In order for TGF.beta.2 to effectively interact with the type II TGF.beta. receptor, the type III receptor must also be present (Derynck (1994) TIBS 19:548-553). Vascular endothelial cells lack the type III receptor. Instead endothelial cells express a structurally related protein called endoglin (Cheifetz et al., (1992) J. Biol. Chem. 267:19027-19030), which only binds TGF.beta.1 and TGF.beta.3 with high alnnity. Thus, the relative potency of the TGF.beta.'s reflect the type of receptors expressed in a cell and organ system.
In addition to the regulation of the components in the multifactorial signaling pathway, the distribution of the synthesis of TGF.beta. polypeptides also affects physiological function. The distribution of TGF.beta.2 and TGF.beta.3 is more limited (Derynck et al., (1988) EMBO J 7:3737-3743) than TGF.beta.1, e.g., TGF.beta.3 is limited to tissues of mesenchymal origin, whereas TGF.beta.1 is present in both mesenchymal and epithelial cells.
TGF.beta.1 is a multifunctional cytokine critical for tissue repair. High concentrations of TGF.beta.1 are delivered to the site of injury by platelet granules (Assoian and Sporn, (1986) J Cell Biol. 102:1217-1223.). TGF.beta.1 initiates a series of events that promote healing including chemotaxis of cells such as leukocytes, monocytes and fibroblasts, and regulation of growth factors and cytokines involved in angiogenesis, cell division associated with tissue repair and inflammatory responses. TGF.beta.1 also stimulates the synthesis of extracellular matrix components (Roberts et aL, (1986) Proc. Natl. Acad Sci USA 83:4167-4171; Sporn et al., (1983) Science 219:1329-1330; Massague, (1987) Cell 49:437-438) and most importantly for understanding the pathophysiology of TGF.beta.1, TGF.beta.1 autoregulates its own synthesis (Kim et al., (1989) J Biol Chem 264:7041-7045).
A number of diseases have been associated with TGF.beta.1 overproduction. Fibrotic diseases associated with TGF.beta.1 overproduction can be divided into chronic conditions such as fibrosis of kidney, lung and liver and more acute conditions such as dermal scarring and restenosis. Synthesis and secretion of TGF.beta.1 by tumor cells can also lead to immune suppression such as seen in patients with aggressive brain or breast tumors (Arteaga et al., (1993) J Clin Invest 92: 2569-2576). The course of Leishmanial infection in mice is drastically altered by TGF.beta.1 (Barral-Netto et al., (1992) Science 257:545-547). TGF.beta.1 exacerbated the disease, whereas TGF.beta.1 antibodies halted the progression of the disease in genetically susceptible mice. Genetically resistant mice became susceptible to Leishmanial infection upon administration of TGF.beta.1.
The profound effects of TGF.beta.1 on extracellular matrix deposition have been reviewed (Rocco and Ziyadeh, (1991) in Contemporary Issues in Nephrology v23, Hormones, autocoids and the kidney. ed. Jay Stein, Churchill Livingston, New York pp391-410; Roberts et al., (1988) Rec. Prog. Hormone Res. 44:157-197) and include the stimulation of the synthesis and the inhibition of degradation of extracellular matrix components. Since the structure and filtration properties of the glomerulus are largely determined by the extracellular matrix composition of the mesangium and glomerular membrane, it is not surprising that TGF.beta.1 has profound effects on the kidney. The accumulation of mesangial matrix in proliferative glomerulonephritus (Border et al., (1990) Kidney Int. 37:689-695) and diabetic nephropathy (Mauer et al., (1984) J. Clin Invest.74:1143-1155) are clear and dominant pathological features of the diseases. TGF.beta.1 levels are elevated in human diabetic glomerulosclerosis (advanced neuropathy) (Yamamoto et al., (1993) Proc. Natl. Acad. Sci. 90:1814-1818). TGF.beta.1 is an important mediator in the genesis of renal fibrosis in a number of animal models (Phan et al., (1990) Kidney Int. 37:426; Okuda et al., (1990) J. Clin Invest. 86:453). Suppression of experimentally induced glomerulonephritus in rats has been demonstrated by antiserum against TGF.beta.1 (Border et al., (1 990) Nature 346:371) and by an extracellular matrix protein, decorin, which can bind TGF.beta.1 (Border et al., (1992) Nature 360:361-363).
Too much TGF.beta.1 leads to dermal scar-tissue formation. Neutralizing TGF.beta.1 antibodies injected into the margins of healing wounds in rats have been shown to inhibit scarring without interfering with the rate of wound healing or the tensile strength of the wound (Shah et al, (1992) Lancet 339:213-214). At the same time there was reduced angiogenesis, reduced number of macrophages and monocytes in the wound, and a reduced amount of disorganized collagen fiber deposition in the scar tissue.
TGF.beta.1 may be a factor in the progressive thickening of the arterial wall which results from the proliferation of smooth muscle cells and deposition of extracellular matrix in the artery after balloon angioplasty. The diameter of the restenosed artery may be reduced 90% by this thickening, and since most of the reduction in diameter is due to extracellular matrix rather than smooth muscle cell bodies, it may be possible to open these vessels to 50% simply by reducing extensive extracellular matrix deposition. In uninjured pig arteries transfected in vivo with a TGF.beta.1 gene, TGF.beta.1 gene expression was associated with both extracellular matrix synthesis and hyperplasia (Nabel et al., (1993) Proc. Natl. Acad. Sci USA 90-10759-10763). The TGF.beta.1 induced hyperplasia was not as extensive as that induced with PDGF-BB, but the extracellular matrix was more extensive with TGF.beta.1 transfectants. No extracellular matrix deposition was associated with FGF-1 (a secreted form of FGF) induced hyperplasia in this gene transfer pig model (Nabel (1993) Nature 362:844-846).
There are several types of cancer where TGF.beta.1 produced by the tumor may be deleterious. MATLyLu rat cancer cells (Steiner and Barrack, (1992) Mol. Endocrinol. 6:15-25) and MCF-7 human breast cancer cells (Arteaga et al., (1993) Cell Growth and Differ. 4:193-201) became more tumorigenic and metastatic after transfection with a vector expressing the mouse TGF.beta.1. In breast cancer, poor prognosis is associated with elevated TGF.beta. (Dickson et al., (1987) Proc. Natl. Acad Sci. USA 84:837-841; Kasidetal., (1987) Cancer Res. 47:5733-5738; Daly et al., (1990) J Cell Biochem 43:199-21 1; Barrett-Lee et aL, (1990) Br. J Cancer 61:612-617; King et al., (1989) J Steroid Biochem 34:133-138; Welch et al., (1990) Proc. Natl. Acad Sci. 87:7678-7682; Walker et aL., (1992) Eur J Cancer 238: 641-644) and induction of TGF.beta.1 by tamoxifen treatment (Butta et al., (1992) Cancer Res 52:4261-4264) has been associated with failure of tamoxifen treatment for breast cancer (Thompson et al., (1991) Br. J Cancer 63:609-614). Anti TGF.beta.1antibodies inhibit the growth of MDA-231 human breast cancer cells in athymic mice (Arteaga et al., (1993) J Clin Invest 92: 2569-2576), a treatment which is correlated with an increase in spleen natural killer cell activity. CHO cells transfected with latent TGF.beta.1 also showed decreased NK activity and increased tumor growth in nude mice (Wallick et al., (1990) J Exp Med 172:1777-1784). Thus, TGF.beta.1 secreted by breast tumors may cause an endocrine immune suppression.
High plasma concentrations of TGF.beta.1 have been shown to indicate poor prognosis for advanced breast cancer patients (Anscher et al. (1993) N Engl J Med 328:1592-8). Patients with high circulating TGF.beta. before high dose chemotherapy and autologous bone marrow transplantation are at high risk for hepatic veno-occlusive disease (15-50% of all patients with a mortality rate up to 50%) and idiopathic interstitial pneumonitis (40-60% of all patients). The implication of these findings is 1) that elevated plasma levels of TGF.beta.1can be used to identify at risk patients and 2) that reduction of TGF.beta.1 could decrease the morbidity and mortality of these common treatments for breast cancer patients.
PDGF
Platelet-derived growth factor (PDGF) was originally isolated from platelet lysates and identified as the major growth-promoting activity present in serum but not in plasma. Two homologous PDGF isoforms have been identified, PDGF A and B, which are encoded by separate genes (on chromosomes 7 and 22). The most abundant species from platelets is the AB heterodimer, although all three possible dimers (AA, AB and BB) occur naturally. Following translation, PDGF dimers are processed into .apprxeq.30 kDa secreted proteins. Two cell surface proteins that bind PDGF with high affinity have been identified, .alpha. and .beta. (Heldin et al., Proc. Natl. Acad. Sci., 78: 3664 (1981); Williams et al., Proc. Natl. Acad. Sci., 79: 5867 (1981)). Both species contain five immunoglobulin-like extracellular domains, a single transmembrane domain and an intracellular tyrosine kinase domain separated by a kinase insert domain. The functional high affinity receptor is a dimer and engagement of the extracellular domain of the receptor by PDGF results in cross-phosphorylation (one receptor tyrosine kinase phosphorylates the other in the dimer) of several tyrosine residues. Receptor phosphorylation leads to a cascade of events that results in the transduction of the mitogenic or chemotactic signal to the nucleus. For example, in the intracellular domain of the PDGF .beta. receptor, nine tyrosine residues have been identified that when phosphorylated interact with different src-homology 2 (SH2) domain-containing proteins including phospholipase C-g, phosphatidylinositol 3'-kinase, GTPase-activating protein and several adapter molecules like Shc, Grb2 and Nck (Heldin, Cell, 80: 213 (1995)). In the last several years, the specificities of the three PDGF isoforms for the three receptor dimers (.alpha..alpha., .alpha..beta., and .beta..beta.) has been elucidated. The a-receptor homodimer binds all three PDGF isoforms with high affinity, the .beta.-receptor homodimer binds only PDGF BB with high affinity and PDGF AB with approximately 10-fold lower affmity, and the .alpha..beta.-receptor heterodimer binds PDGF BB and PDGF AB with high affinity (Westermark & Heldin, Acta Oncologica, 32: 101 (1993)). The specificity pattern results from the ability of the A-chain to bind only to the a-receptor and of the B-chain to bind to both .alpha. and .beta.-receptor subunits with high affinity.
The earliest indication that PDGF expression is linked to malignant transformation came with the finding that the amino acid sequence of the PDGF-B chain is virtually identical to that of p28.sup.sis, the transforming protein of the simian sarcoma virus (SSV) (Waterfield et al. Nature, 304: 35 (1983); Johnsson et al., EMBO J., 3: 921 (1984)). The transforming potential of the PDGF-B chain gene and, to a lesser extent, the PDGF-A gene was demonstrated soon thereafter (Clarke et al., Nature, 308: 464 (1984); Gazit et al., Cell, 39: 89 (1984); Beckmann et al., Science, 241: 1346; Bywater et al., Mol. Cell. Biol., 8: 2753 (1988)). Many tumor cell lines have since been shown to produce and secrete PDGF, some of which also express PDGF receptors (Raines et al., Peptide Growth Factors and Their Receptors, Springer-Verlag, Part I, p 173 (1990)). Paracrine and, in some cell lines, autocrine growth stimulation by PDGF is therefore possible. For example, analysis of biopsies from human gliomas has revealed the existence of two autocrine loops: PDGF-B/.beta.-receptor in tumor-associated endothelial cells and PDGF-A/.alpha.-receptor in tumor cells (Hermansson et al., Proc. Natl. Acad. Sci., 85: 7748 (1988); Hermansson et al., Cancer Res., 52: 3213 (1992)). The progression to high grade glioma was accompanied by the increase in expression of PDGF-B and the .beta.-receptor in tumor-associated endothelial cells and PDGF-A in glioma cells. Increased expression of PDGF and/or PDGF receptors has also been observed in other malignancies including fibrosarcoma (Smits et al., Am. J. Pathol., 140: 639 (1992)) and thyroid carcinoma (Heldin et al., Endocrinology, 129: 2187 (1991)).
In view of its importance in proliferative disease states, antagonists of PDGF may find usefilm clinical applications. Currently, antibodies to PDGF (Johnsson et al., (1985) Proc. Natl. Acad. Sci., U. S. A. 82: 1721-1725; Ferns et al., (1991) Science 253: 1129-1132; Herrenet al., (1993) Biochimica et Biophysica Acta 1173, 194-302) and the soluble PDGF receptors (Herrenet al., (1993) Biochimica et Biophysica Acta 1173: 294-302; Duanet al., (1991) J. Biol. Chem. 266: 413-418; Tiesman et al., (1993) J. Biol. Chem. 268: 9621-9628) are the most potent and specific antagonists of PDGF. Neutralizing antibodies to PDGF have been shown to revert the SSV-transformed phenotype (Johnsson et al., (1985) Proc. Natl. Acad. Sci. U.S.A. 82: 1721-1725) and to inhibit the development of neointimal lesions following arterial injury (Ferns et al., (1991) Science 253: 1129-1132). Other inhibitors of PDGF such as suramin (Williams et al., (1984) J. Biol. Chem. 259: 5287-5294; Betsholtz et al., (1984) Cell 39 447-457), neomycin (Vassbotn et al., (1992) J. Biol. Chem. 267 15635-15641) and peptides derived from the PDGF amino acid sequence (Engstrom et al., 1992) J. Biol. Chem. 267: 16581-16587) have been reported, however, they are either too toxic or lack sufficient specificity or potency to be good drug candidates. Other types of antagonists of possible clinical utility are molecules that selectively inhibit the PDGF receptor tyrosine kinase (Buchdunger et al., (1995) Proc. Natl. Acad. Sci., U.S.A. 92: 2558-2562; Kovalenko et al, (1994) Cancer Res. 54: 6106-6114).
hKGF
a) Biochemical Properties of hKGF
Human Keratinocyte Growth Factor (hKGF) is a small (26-28KD) basic heparin-binding growth factor and a member of the FGF family. hKGF is a relatively newly identified molecule, which is also known as FGF-7 (Finch et al., (1989) Science 245:752-755). It is a growth factor specific for epithelial cells (Rubin et al., (1989) Proc Natl Acad Sci USA 86:802-806), and its main function is in development/morphogenesis (Werner et al., (1994) Science 266:819-822) and in wound healing (Werner et al., (1992) Proc Natl Acad Sci USA 89:6896-6900). The major in vivo source of hKGF is stromal fibroblasts (Finch et al., (1989) Science 245:752-755). Microvascular endothelial cells (Smola et al., (1993) J Cell Biol 122:417-429) and very recently, activated intraepithelial gd T cells (Boismenu et al., (1994) Science 266:1253-1255) have also been shown to synthesize hKGF. hKGF expression is stimulated in wounds (Werner et al., (1992) Proc Natl Acad Sci USA 89:6896-6900). Several cytokines are shown to be hKGF inducers (Brauchle et al., (1994) Oncogene 9:3199-3204), with IL-1 the most potent one (Brauchle et al., (1994) Oncogene 9:3199-3204; Chedid et al., (1994) J Biol Chem 269:10753-10757). Unlike bFGF, hKGF has a signal peptide and thus is secreted by producing cells (Finch et al., (1989) Science 245:752-755). hKGF can be overexpressed in E. coli and the recombinant protein (.about.19-21 KD) is biologically active (Ron et al., (1993) J Biol Chem 268:2984-2988). The E. coli derived recombinant protein is 10 times more mitogenic than the native protein (Ron et al., (1993) J Biol Chem 268:2984-2988). This difference may be due to glycosylation. The native protein has a potential Asn glycosylation site (Ron et al., (1993) J Biol Chem 268:2984-2988).
The hKGF bioactivity is mediated through a specific cell surface receptor (Miki et al., (1991) Science 251:72-75). The hKGF receptor is a modified FGF receptor resulting from alternative splicing of the C-terminal extracellular region of the FGF-R2 (Miki et al., (1992) Proc Natl Acad Sci USA 89:246-250). NIH/3T3 cells transfected with the hKGF receptor express high affinity (-200 pM) binding sites for hKGF (Miki et al., (1992) Proc Natl Acad Sci USA 89:246-250). The approximate number of specific binding sites per NIH/3T3 cell is about 500,000 (D. Bottaro and S. Aaronson, personal communication). The hKGF receptor binds hKGF and aFGF with similar affinities, and bFGF with about 20 fold less affinity (Miki et al., (1991) Science 251:72-75; Miki et al., (1992) Proc Natl Acad Sci USA 89:246-250). A variant ofthe hKGF receptor has been found to be an amplified gene (i.e., one gene, multiple copies), designated K-SAM, in a human stomach carcinoma cell line (Hattori et al., (1990) Proc Natl Acad Sci USA 87: 5983-5987).
Heparin has been reported to be an inhibitor of hKGF bioactivity (Ron et al., (1993) J Biol Chem 268:2984-2988). This is in contrast to the agonistic effect of heparin for aFGF (Spivak-Kroixman et al., (1994) Cell 79:1015-1024).
b) Role of hKGF in Human Disease
The recombinant hKGF molecule has been available only since 1993. Therefore, there is limited information on the role of hKGF in human disease. The published literature, however, contains evidence that strongly suggests a role for hKGF in at least two human diseases, namely psoriasis and cancer. hKGF has also been implicated in inflammatory bowel disease (P. Finch, personal communication).
Psoriasis
Psoriasis is a skin disorder which can be debilitating (Greaves et al., (1995) N Eng J Medicine 332: 581-588), characterized by hyperproliferation of the epidermis and incomplete differentiation of keratinocytes, together with dermal inflammation (Abel et al., (1994) Scientific American Medicine III-1 to III-18; Greaves et al., (1995) N Eng J Medicine 2:581-588). There is not yet an effective treatment for psoriasis (Anonymous, (1993) Drug & Market Development 4:89-101; Abel et al., (1994) Scientific American Medicine III-1 to III-18; Greaves et al., (1995) N Eng J Medicine 2:581-588). Psoriasis occurs in 0.5 to 2.8 percent of the population with the highest incidence in Scandinavia. In the US in 1992, it was estimated that 4-8 million people affected with psoriasis spent about $600 million for various drugs and related therapies, none of which is very effective. Most of the expenditure was made by about 400,000 patients with severe psoriasis spending $1,000-1,500 annually on treatment. There are about 200,000 new cases of psoriasis every year.
The basic cause of the disorder is not known, but it results from a primary or secondary defect in the mechanisms that regulate epidermal keratinocyte cell division (Abel et al., (1994) Scientific American Medicine III-1 to III-18). Psoriasis responds to steroids and cyclosporine and in that sense is characterized as an immune disease (Abel et al., (1994) Scientific American Medicine III-1 to III-18). Since hKGF is the primary specific growth factor for keratinocytes, its overexpression and deregulation are primary candidates as the cause of for, keratinocyte hyperproliferation in psoriasis. The demonstration that the immune system is a prime regulator of hKGF release (Boismenu et al., (1994) Science 266: 1253-1255; Brauchle et al., (1994) Oncogene 9: 3199-3204; Chedid et al., (1994) J Biol Chem 269: 10753-10757) strengthens the notion that hKGF deregulation is the cause of psoriasis. Furthermore, application of hKGF in porcine wounds creates a histological appearance resembling psoriasis (Staiano-Coico et al., (1993) J Ex Med 178:865-878); keratinocyte derived hKGF in transgenic mice causes pathology reminiscent to psoriasis (Guo et al., (1993) EMBO J 12: 973-986); in situ hybridization experiments demonstrated a moderate and a strong upregulation of hKGF and hKGF receptors respectively in psoriasis (P. Finch, personal communication). In situ hybridization experiments also demonstrated involvement of hKGF in another immune disease namely, inflammatory bowel disease (P. Finch, personal communication).
Cancer
It is well established in the literature that deregulation of the expression of growth factors and growth factor hKGF and/or its receptor is expected to be the transformation event in some human cancers. The transforming ability of the hKGF system has been demonstrated in vitro (Miki et al., (1991) Science 251:72-75). In another study, carcinoma cell-lines have been found to express the hKGF receptor and to respond to hKGF but not to aFGF, while sarcoma cell-lines do not express hKGF receptors and respond to aFGF but not to hKGF (Ishii et al., (1994) Cancer Res 54:518-522).
Gastrointestinal Cancer
Several poorly differentiated stomach cancers have an amplified gene, designated K-sam, which is an isoform of the hKGF-receptor (Katoh et al., (1992) Proc Natl Acad Sci USA 89:2960-2964). In vivo administration of hKGF to rats causes proliferation of pancreatic ductal epithelial cell (Yi et al., (1994) Am J Pathol 145:80-85), hepatocytes, and epithelial cells throughout the gastrointestinal tract (Housley et al., (1994) J Clin Invest 94:1764-1777).
Lung Cancer
Administration of hKGF to rats causes type II pneumocyte hyperplasia similar to the bronchoalveolar cell variant of lung carcinoma (Ulich et al., (1994) J Clin Invest 93:1298-1306).
Breast Cancer
In vivo, hKGF causes mammary duct dilation and rampant epithelial hyperplasia, both of which are common features of breast cancers (Ulich et al., (1994) Am J Pathol 144:862-868; Yi et al., (1994) Am J Pathol 145:1015-1022). However, the ductal epithelium of breastfeeding rats is resistant to the growth promoting effects of hKGF and this is of interest in regard to epidemiological observations that pregnancy in women decreases susceptibility to breast cancer and that dairy cows almost never develop breast cancer (Kuzma, 1977, Breast in Pathology, Mosby Co.). There is additional supporting evidence implicating hKGF in breast cancer. hKGF mRNA has been detected recently in normal human breast tissue and in 12 of 15 breast tumor samples tested (Koos et al., (1993) J Steroid Biochem Molec Biol 45:217-225). The presence of hKGF mRNA in breast tumors considered in conjunction with the observation that hKGF is present in nonneoplastic mammary glands and that hKGF causes rampant proliferation of mammary epithelium suggests that hKGF may be an autocrine or paracrine growth factor important in the regulation of the growth of normal and neoplastic mammary epithelium (Ulich et al., (1994) Am J Pathol 144:862-868). Infiltrating ductal mammary adenocarcinoma is characteristically enveloped by a desmoplasmic stroma that has been postulated to represent a defensive host response to the carcinoma (Ulich et al., (1994) Am J Pathol 144:862-868). Since hKGF is stroma derived it is possible that the desmoplasmic stroma contributes rather than inhibits the growth of the tumor.
Prostate Cancer
The growth promoting effect of androgens on prostate tumors appears to be mediated through hKGF (Yan et al., (1992) Mol Endo 6:2123-2128), as androgens induce the expression of hKGF in prostate stroma cells. Prostate tumors that are androgen dependent in vivo, are androgen independent in vitro, but hKGF dependent (Yan et al., (1992) Mol Endo 6:2123-2128). In agreement with the role of hKGF as andromedin is the observation that hKGF functions in epithelial induction during seminal vesicle development, a process that is directed by androgen (Alarid et al., (1994) Proc Natl Acad Sci USA 91:1074-1078). Furthermore, hKGF causes aberrant activation of the androgen receptor, thus probably contributing to the failure of androgen ablation therapy in prostate cancer (Culig et al., (1994) Cancer Res 54:5474-5478). Based on this information, it is possible that genetic alterations cause hKGF to escape androgen regulation and thus convert the androgen dependent tumor into an androgen independent, highly malignant tumor. Such tumors would still be able to express the androgen regulated marker PSA, as hKGF also causes the aberrant activation of the androgen receptor. It is also likely that hKGF might be responsible for Benign Prostate Hypertrophy (BPH), a common health problem in older men (D. Bottaro, personal communication).
d) hKGF Competitors
To date, a monoclonal antibody and a short hKGF-receptor derived peptide (25-mer) have been described as hKGF competitors (Bottaro et al., (1993) J Biol Chem 268:9180-9183). The monoclonal antibody, designated 1G4, has a Kd of 200 pM for hKGF. The short peptide inhibits hKGF binding to the cell surface of NIH/3T3 cells expressing the human receptor with a Ki of about 1-5 .mu.M. Bottaro et al. (WO 94/25057) provide hKGF-receptor peptides which inhibit binding between hKGF and its receptor. Also provided is a method of assaying test compounds for the ability to inhibit hKGF receptor-mediated cell proliferation.
e) Assaying for Receptor-Growth Factor Interaction
Blocking the interaction of growth factors and lymphokines with their cell surface receptor using antagonists has been an approach for disease treatment. The discovery of such antagonists requires the availability of biochemical assays for the receptor-growth factor or lymphokine interaction. A classic assay has been the competitive inhibition of radiolabeled growth factor or lymphokine (tracer) to its cell surface receptor. These types of assays utilize cell lines that express the relevant receptor on their surface and determines the amount of cell bound tracer in the presence of various concentrations of potential antagonists. Additionally, other assays utilize membrane extracts from cell lines that express the relevant receptor, and tracer binding is followed by filter binding (see Nenquest Drug Discovery System: Human Tumor Necrosis Factor-Alpha, NEN Research Products, E. I. DuPont de Nemours & Co. (Inc.), Boston, Mass.) or by immobilizing the membrane extracts onto solid supports (Urdal et al., (1988) J Biol Chem 263:2870-2877; Smith et al., (1991) Bioch Bioph Res Comm 176:335-342). Receptor induced electrophoretic mobility shift of tracer has been applied to identify the presence and size of cell surface receptors by crosslinking the receptor to the tracer and then analyzing on denaturing gels (for example see Kull et al., (1985) Proc natl Acad Sci USA 82:5756-5760; Hohmann et al., (1989) J Biol Chem 264:14927-14934; Stauber et al., (1989) J Biol Chem 264:3573-3576). The use of native gels and non-crosslinked complexes has not been described for growth factors or lymphokines and their receptors, but has been widely applied to study nucleic acid protein interactions (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
Screening of various cancer cell lines for the presence of hKGF receptors by PCR, revealed that all carcinoma cell lines express hKGF receptor MRNA while sarcoma cell lines do not. The presence of MRNA does not necessarily mean that hKGF receptor will be present on the surface of these cells. For hKGF, only cell based assays have been described using Balb/MK keratinocytes (Weissman, (1983) Cell 32: 599-606) or NIH/3T3 cells transfected with the hKGF receptor (Miki, (1992) Proc. Natl. Acad. Sci. USA 89:246-250).
SELEX
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in United States patent application Ser. No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, United States patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,475,096, United States patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Methods for Identifying Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplfing the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, United States patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. United States patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands" describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. United States patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed "Counter-SELEX." United States patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX," now abandoned (see U.S. Pat. No. 5,567,588), describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. United States patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled "Nucleic Acid Ligands to HIV-RT and HIV-1 Rev," now issued as U.S. Pat. No. 5,496,938, describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. United States patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX," now issued as U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in United States patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," now abandoned (see, U.S. Pat. No. 5,660,985), that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. United States patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). United States patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2'-Modified Nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide fimctional units as described in United States patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX", now U.S. Pat. No. 5,637,459, and United States patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX," now U.S. Pat. No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.